Project summary STING-mediated type I interferon (IFN) signaling plays an important role in innate immune response in infection and autoimmune disease. The STING signaling pathway is unique in that it requires STING trafficking from the ER through ERGIC/Golgi to vesicles, during which time STING recruits kinase TBK1 and transcription factor IRF3 which subsequently activates IFN gene expression. STING trafficking also activates other non-IFN pathways under pathophysiological conditions such as cell death and autophagy, although the mechanism is unclear. We showed that dominant STING gain-of-function mutations constitutively activate STING trafficking in vitro and causes lung disease and T cell cytopenia independently of IFN signaling in vivo. In preliminary studies we performed a time-resolved proteomic study using STING-APEX2 and identified several cofactors at various membrane locations along the trafficking route. Our main hypothesis is that STING trafficking is a primordial way for STING to regulate multiple cellular processes including IFN and non-IFN pathways, such as calcium signaling, cell death and autophagy. The non-IFN functions of STING are likely important for human physiology because at least one such function is implicated in human disease STING-associated vasculopathy with onset in infancy (SAVI). We will study the function of STING trafficking and non-IFN functions through the following three specific aims: In Aim 1, we will study the cellular process of STING trafficking by discovering new cofactors along the trafficking route and elucidating their mechanism of action. In Aim 2, we will investigate one particular non-IFN function, the UPR, that is mediated by STING trafficking in T cells, using the dominant gain-of-function Sting- N153S mouse with active disease pathology. In Aim 3, we will broadly explore non-IFN functions of STING in a variety of physiological conditions, using a new point mutant mouse Sting-S365A, which selectively blocks STING-mediated IFN signaling while retaining normal trafficking and other signaling capabilities. Studies proposed here will address a previously understudied but important area of STING biology and reveal new functions of STING with pathophysiological relevance.